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rilE  LFXITHANS 


WALDEMAR  KOCH 


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rOURDED  BY  JOHN  D.  BOCEEFELLER 


The  Decennial  Publications 


THE   LECITHANS 

THEIK  FUNCTION  IN  THE  LIFE  OF  THE  CELL 

BY 

WALDEMAR    KOCH 

ASSISTANT  IN  PHAEMACOLOOT 


PRINTED  FROM  VOLUME  X 


CHICAGO 

THE  UNIVERSITY  OF  CHICAGO  PRESS 

1902 


K11 


Copyright  1902 

BY  THE  UNIVEESITY  OF  CHICAGO 


PRINTED  NOVEMBEH  1, 1902 


THE  LECITHANS 

THEIR  FUNCTION  IN  THE  LIFE  OF  THE  CELL 

Waldemar  Koch 

The  ash  left  on  the  incineration  of  tissues  obtained  from  various  parts  of  the  body, 
especially  the  brain,  has  long  been  known  to  contain  phosphorus.  Of  the  chemical 
combination  in  which  this  phosphorus  was  present  in  the  original  tissues  nothing  was 
known  until  Gobley'  carefully  studied  an  organic  phosphorus-containing  body,  which 
he  isolated  from  eggs  and  called  lecithin.  He  obtained  as  splitting  products  glycero- 
phosphoric  acid  and  some  of  the  fatty  acids.  Diaconow^  continued  this  work  at  the 
suggestion  of  Hoppe  Seyler,  and  isolated  as  splitting  products  glycerophosphoric  acid, 
stearic  and  oleic  acids,  and  a  base  which  he  identified  with  Baeyer's  neurin  and  the 
neurin  obtained  by  Liebreich''  from  his  protagon  by  decomposition  with  barium 
hydrate.  From  the  ease  with  which  his  lecithin  could  be  split  up  Diaconow  con- 
cluded that  it  was  a  neurin  salt  of  distearyl  glycerophosphoric  acid.  This  view  was, 
however,  disproved  by  Hundeshagen^  on  account  of  the  fact  that  the  body  prepared  by 
the  union  of  neurin  and  distearyl  glycerophosphoric  acid  in  alcohol  solution  would  not 
give  the  characteristic  myelin  forms,  although  it  possessed  all  the  other  properties  of 
lecithin.  Strecker*^  brought  confusion  into  this  subject  by  identifying  the  base 
obtained  by  him  from  lecithin  with  the  cholin  he  had  isolated  from  bile.°  Thudichum' 
pointed  out  the  difference  between  the  body  derived  from  bile  and  the  base  isolated 
from  lecithin,  and  identified  the  latter  as  neurin  by  a  number  of  analyses  made  with 
carefully  purified  material.  In  the  book  above  referred  to  Thudichum  also  records 
some  other  important  observations.  Among  the  large  number  of  compounds  isolated 
by  him  from  the  brain  there  are  some  which  do  not  contain  glycerin;  as  he  always 
finds  phosphorus  in  the  form  of  orthophosphoric  acid,  he  concludes  to  call  these 
bodies  "  Pho.sphatids "  and  consider  them  rather  as  derivatives  of  orthophosphoric 
acid  than  glycerophosphoric  acid,  as  previously  accepted.  His  formulse  resemble  the 
types  of  the  Typentheorie  of  Gerhardt  and  Wurtz,  in  leaving  the  exact  building  up 
of  the  molecule  a  matter  of  doubt.  From  a  study  of  the  fatty  acids  in  his  various 
compounds  Thudichum  concludes,  contrary  to  Diaconow,  that  there  are  no  i^hosphatids 
with  only  one  fatty  acid,  but  that  each  one  contains  either  palmitic,  stearic,  or  mar- 

I  Gobley,  Journal  de  pharmacie  et  de  chimie  (1850),  « Hdndeshaqen, /oumaI/«r  profc««c7ie  CAemie  (1SS3), 

Vol.  XVII,  p.  401,  and  Vol.  XVIII,  p.  107.  Vol.  XXVIII,  p.  219. 

^Diaconow,  Hoppe  SeyJers  medicinUch^hemische  Un-  sStreckee,  Liebig-,  Annalen  der  Chemie  und  Fhar- 

tersuchungen,lS66,\o\.Il,  p.  221;  also  Centralblatt  far  die  macie  (186S)   CXLVIII  p  18 
medicinischen    WUsenschaften  (1868),  Vol.  VI,    pp.  2,  97,  '  ■  P.      • 

and  434.  '  Ibid.  (1862),  Vol.  CXSIII,  p.  353. 

'LiEBREICH,  Licbig's  Annalen  der  Chemie  und  Fhar-  'Thttbichcm,  Die  chcmische  Konstitution  des  GeMms 

macie  (1865),  Vol.  CXXXIV,  p.  34.  des  Menschen  und  der  Tiere,  1901.  p.  123. 

93 


The  Leoithans 


gearic  as  one  of  the  constituents;  which,  however,  gives  no  character  to  the  molecule, 
while  the  other  acid  —  oleic  for  brain  lecithin,  kephalinic  for  kephalin — gives  to  the 
molecule  its  distinctive  properties. 

Without  considering  further  the  important  question  of  the  structure  of  these  com- 
pounds, I  would  propose  to  classify  them  under  the  general  term  "Lecithans."  The 
introduction  of  the  word  "  lecithan  "  as  a  group  name  seems  preferable  to  the  use  of 
an  entirely  new  and  unfamiliar  term  like  "  phosphatids,"  as  proposed  by  Thudichum. 
At  the  same  time,  the  change  of  the  last  syllable  of  lecithin  to  an  gives  sufficient 
variation  to  prevent  any  such  confusion  as  attended  the  generalization  of  the  word 
"albumen."  The  lecithans,  then,  are  substances  containing  in  the  molecule  phos- 
phoric acid,  fatty  acids,  nitrogen,  and,  in  most  cases,  glycerin.  They  resemble  each 
other  very  closely  in  their  physical  appearance,  being  waxy,  non-crystalline,  and  very 
hygroscopic.  Toward  water  they  all  show  the  same  behavior,  although  their  solubility 
or  the  solubility  of  their  salts  in  organic  solvents  may  vary. 

The  very  general  distribution  of  the  lecithans  in  all  forms  of  living  tissues  speaks 
for  their  value  in  the  life  of  the  cell.  A  more  careful  study  of  these  compounds  indi- 
cates that  they  are  valuable  in  two  ways:  first,  on  account  of  their  physical  properties; 
and,  secondly,  on  account  of  their  chemical  behavior. 

PHYSICAL    PEOPEKTIES 

The  behavior  of  the  lecithans  with  water  seems  of  especial  interest,  and  can  be 
watched  under  the  microscope.  A  waxy  piece  of  brain  lecithin  placed  in  water  first 
swells  up  and  then  gives  off  long  filaments  (called  myelins)  which  sometimes  resemble 
a  shepherd's  crook,  at  other  times  a  mass  of  twisted  skein.  If  allowed  to  stand  for 
some  time,  with  frequent  shaking,  a  perfect  emulsion  is  finally  formed.  A  lecithan 
which  has  been  part  of  the  living  tissues,  such  as  brain  lecithin,  gives  a  much  more  perfect 
emulsion  than  egg  lecithin,  which  is  merely  stored  food  material.  If  such  an  emul- 
sion is  the  substratum  of  the  living  cell — and  there  seems  good  reason  to  consider  it 
so  — it  may  explain  some  of  the  physical  properties  of  living  protoplasm.  The  study 
of  this  emulsion  is  especially  interesting  in  connection  with  the  changes  in  the  physical 
conditions  of  the  living  cell  brought  about  by  electrolytes,  as  shown  by  the  recent 
work  of  J.  Loeb  and  his  school. 

Action  of  electrolytes  on  an  emulsion  of  brain  lecithin. — Four  g.  of  brain  lecithin 
(free  from  calcium,  and  containing  less  than  1  per  cent,  sodium  or  potassium) 
are  treated  with  one  liter  distilled  water.  The  resulting  emulsion  is  sufficiently  trans- 
parent for  purposes  of  study,  can  be  filtered  unchanged,  has  a  neutral  reaction  to 
litmus,  and  remains  unaltered  for  weeks,  especially  after  sterilization  by  boiling.  The 
results  of  my  experiments  with  this  emulsion  may  be  classified  as  follows: 

Univalent  kations. —  Salts  of  Na,  K,  NH4,  Li,  Ag,  even  in  very  concentrated  solu- 
tion, give  no  precipitate  and  have  apparently  no  effect  on  the  emulsion.  The  hydro- 
gen ion  is  an  exception,  in  the  case  of  acids  which  are  sufficiently  dissociated.     A 

94 


Waldemar  Koch 


concentration  of  ^ttk  sulphuric  will  give  a  precipitate.     Carbonic  acid  is  not  BuflSciently 

soluble  to  give  any  precipitate. 

Divalent  kations.—Mg,  Ca,  Sr,  Ba,  Co,  Ni,  Fe",  Zn,  Cd,  Cu,  and  Pb  all  give  a 
precipitate  which,  similar  to  the  one  with  acids,  is  flocculent,  gelatinous,  and  settles  to 
the  bottom  in  less  than  one  hour,  leaving  the  supernatant  liquid  perfectly  clear.  The 
concentrations  which  will  just  give  the  precipitate  vary  somewhat  and  have  been 

found  in  the  case  of  Ca,  Sr,  and  Ba  to  be  jrr^  ,  tt:  ,  and  stj  j  respectively. 

Trivalent  kations. —  Fe'",  Al,  Au  give  no  precipitate  and  behave  like  monovalent 
kations.  Cr  gives  unsatisfactory  results.  Au,  after  standing  for  several  hours,  is  pre- 
cipitated in  the  metallic  state. 

Am'ons. —  CI,  Br,  I,  SO4",  oxalate,  citrate,  and  ferrocyanide  (K4Fe(CN)5)  give 
no  precipitate,  and  even  in  concentrated  solution  have  no  apparent  effect  on  the  emul- 
sion.     OH  is  an  exception,  causing  the  emulsion  to  clear  up. 

Non-electrolytes. — Albumins,  peptones,  glucose,  urea,  alkaloids,  and  narcotics  like 
urethan  and  chloral  give  no  precipitation  reactions  and  leave  the  emulsion  apparently 
unchanged.  Chloroform  has  a  tendency  to  be  emulsified  by  the  emulsion,  a  reaction 
which  Thudichum  had  already  observed  with  ether. 

The  precipitations  above  observed  with  the  hydrogen  ion  and  divalent  kations 
seem  to  be  of  an  entirely  physical  nature  because: 

1.  They  are  independent  of  the  concentration  of  the  lecithin.  An  nnnr.  lecithin 
emulsion  will  begin  to  precipitate  with  about  the  same  concentration  of  the  divalent 
kation  as  an  ^j=j7;  emulsion.     Stronger  emulsions  are  not  sufficiently  transparent  for 

observation. 

2.  Removal  of  the  supernatant  liquid  by  decantation  and  the  addition  of  water 
will  cause  the  precipitates  to  redissolve. 

Cadmium,  copper,  and  other  salts  of  various  lecithans  have  been  prepared  in 
alcohol  solution  and  analyzed,  but  they  are  readily  broken  up  on  the  addition  of  water, 
and  belong  to  a  class  of  physical  compounds  even  more  unstable  than  ordinary  double 
salts.  It  would  seem,  then,  that  when  a  certain  limiting  concentration  of  the  divalent 
kation  is  reached,  the  emulsion  can  no  longer  exist  and  the  lecithin  is  precipitated, 
carrying  with  it  possibly  some  of  the  salt.  Very  interesting,  on  account  of  the  possi- 
bility of  furnishing  an  explanation  of  such  results  as  Loeb'  obtains  with  Fundulus,  are 
the  antagonistic  effects  of  univalent  kations  in  preventing  the  precipitation.  Near  the 
limits  at  which  Ca  will  just  give  a  precipitate,  a  very  small  amount  of  Na  will  suffice  to 
prevent  this  precipitate ;  as  more  Ca  is  added,  relatively  more  Na  is  needed.  A  direct 
comparison  of  my  results  with  J.  Loeb's  is  not  possible,  because,  in  the  first  piece,  the 
amounts  of  Ca,  Na,  and  lecithin  in  the  Fundulus  egg  are  not  known,  and,  in  the 
second  place,  the  reaction  between  the  solution  and  the  egg  does  not  come  about  as 

8L0EB,  American  Journal  of  Physiology  (1902),  Vol.  VI,  p.  ill. 

95 


The  Lecithans 


After 
Three  Hours 

-  Immediate  ppt. 

Ppt.  settled 

-  No  ppt. 

No  ppt. 

-  No  ppt. 

No  ppt. 

-  Immediate  ppt. 

Ppt.  settled 

-  No  ppt. 

No  ppt. 

-  No  ppt. 

No  ppt. 

-  No  ppt. 

No  ppt. 

directly  as  in  my  case.     The  following  table  gives  the  data  obtained  with  an  emulsion 
of  brain  lecithin: 

I.  5  CO.  =i^  emul.  +  5  c.c.  water       +    5  c.c.  j^  Ca(N03)2  - 

■5c.c.5mNaCl+    5  c.c.  |^  Ca(N03)2  - 

I  emul.  +  5  c.c.  ^  NaCl  +  1.5  c.c.  ^  Ca(N03)2  - 

emul.  +  5  c.c.  jg  NaCl  +    5  c.c.  jg  Ca(N03)2  - 

emul.  +  5  c.c.  ^  NaCl  +3.5  c.c.  ^  Sr(N03)2   - 

VI.  5  c.c.  ^  emul.  +  5  c.c.  2hn  KC1+    5  c.c.  -  Ca(N03)2  - 

VII.  5  c.c.  ^  emul.  +  5  c.c.  ^  Fed,  +    5  c.c.  ^  Ca(N03)2  - 

VIII.  5  c.c.  qTjp;  emul.  +  5  c.c.  urea  concentrated  solution 

+  5  c.c.:^  Ca(N03)2     Ppt.  formed  slowly    Ppt.  settled 

IX.  5  c.c.  ^^  emul.  +  5  c.c.  glucose  concentrated  solution 

+  5  c.c.  yq  Ca(N03)2    Ppt.  formed  slowly   Ppt.  settled 

We  may  conclude,  then,  that  the  precipitation  of  lecithin  by  divalent  kations  is  a 
physical  phenomenon  probably  of  an  electrical  nature,  because: 

1.  Non-electrolytes  do  not  prevent  the  precipitation  (I,  VIII,  IX). 

2.  The  trivalent  kation  Fe'"  is  much  more  efficient  in  preventing  the  precipita- 
tion than  a  monovalent  one  like  Na  (II,  VII). 

3.  The  precipitate  is  formed  independent  of  the  concentration  of  the  lecithin  and 
can  be  redissolved  by  the  addition  of  water. 

The  application  of  these  observations  to  Loeb's  results  must  be  postponed  until 
other  lecithans  have  been  more  carefully  studied. 

CHEMICAL    PROPERTIES 

The  chemical  properties  of  the  lecithans  depend  on  two  groups  in  the  molecule: 
first,  the  fatty  acids,  and,  second,  the  complex  of  which  the  nitrogen  is  a  part.  The 
phosphoric  acid,  although  the  nucleus  and  very  important  in  the  building  up  of  the 
molecule,  does  not  seem  to  enter  into  any  reaction,  except  on  the  complete  destruction 
of  the  lecithan ;  as  Halliburton^  has  found  the  phosphorus  to  decrease  in  degenerating 
nerves  only  after  the  eighth  day. 

Each  lecithan  contains,  according  to  Thudichum,  two  fatty  acids  in  the  molecule: 
one  —  either  palmitic,  stearic,  or  margaric — does  not  impart  any  particular  property  to 
the  compound;  the  other — oleic  in  the  case  of  lecithin,  kephalinic  in  the  case  of 
kephalin — gives  to  the  molecule  its  distinctive  character.  This  distinctive  group  is 
always  unsaturated,  will  therefore  add  iodine,  and  bring  about  the  reduction  of  osmic 
acid.     Upon  this  group,  then,  depends  the  use  of  osmic  acid  as  a  stain  for  nervous 

9  W.  D.  Hallibueton,  The  Cliemical  Side  of  Nervous  Activity,  1901,  p.  87. 

96 


Waldemar  Koch 


tissues  in  histological  technique.  The  value  of  osmic  acid  as  a  general  test  for  fats 
depends  on  the  fact  that  all  fats  in  the  body  contain  some  oleates.  Pure  stearates  and 
palmitates  will  not  give  the  test.  The  darkening  of  the  lecithans  on  exposure  to  the 
air  is  also  dependent  on  this  group.  In  the  case  of  kephalin,  the  change  on  exposure 
to  the  air  takes  place  so  rapidly  as  to  suggest  an  autoxidizable  substance  capable  of 
activating  oxygen.  The  guiac-blue  reaction,  however,  gives  a  negative  result ;  and 
Thudichum'"  has  shown  that  kephalin  exposed  to  an  atmosphere  of  oxygen  in  a  eudi- 
ometer will  not  decrease  the  volume  of  the  gas.  The  change  is  probably  due  to  an 
internal  rearrangement  in  the  molecule,  and  takes  place  within  the  molecule  of  the 
fatty  acid  itself;  as  Thudichum"  obtained  from  kephalin  an  acid  by  saponification 
(kephalinic  acid)  which  exhibited  the  same  changes  as  the  mother-substance. 

Less  apparent,  but  nevertheless  important,  are  the  changes  which  the  molecules 
of  the  lecithans  undergo  in  the  complex  which  contains  the  nitrogen.  Thus  Hallibur- 
ton'" has  found  the  cholin  to  increase  in  the  cerebro-spinal  fluid  as  the  result  of  general 
paralysis.  For  the  quantitative  investigation  of  the  cholin  or  neurin  the  methyl 
groups  attached  to  the  nitrogen  seem  especially  useful,  as  Herzig  and  Meyer"  have 
devised  a  method  by  which  such  groups  can  be  accurately  determined.  The  descrip- 
tion of  the  method  is  not  easily  accessible.  I  will  therefore  repeat  it  here,  with  such 
modifications  as  have  been  found  useful,  before  going  on  to  describe  the  results 
obtained  with  lecithans  from  various  sources. 


HEEZIG  AND  MEYER  S  DETERMINATION  OF  METHYL  ATTACHED  TO  NITROGEN 

The  apparatus  consists  of  a  double  glass  bulb,  4  cm.  wide  at  the  largest  diameter  and 
2^  cm.  at  the  narrowest  diameter,  and  12  cm.  high.     The  bulb  (a)  is  connected  to  (6)  by 

a  glass  tube  which  runs  to  the  bot- 
tom of  (6).  The  double  bulbs  are 
placed  in  an  iron  sand  bath  with 
double  bottom  and  a  par- 
l  tition  (cd)  so  that  (a)  can 

•^"'/'""^    be  heated  in  sand,  while 
(b)  remains    compara- 
tively cool.      C  is  a  flask  for  catching 
the  distillation  products  from  the  bulbs, 

and  D  is  a  condenser  kept  at  a  tem-      \j ,,, ^^  j^ 

perature  of  from  40°  to  50°  C.    E  is       ""  ^  '' 

a  beaker  into  which  the  water  enters 
at  G,  is  heated  to  a  temperature  of 
from  40°  to  50°  C.  by  a  Bunsen  burner,  Q 
and  is  drawn  off  by  means  of  a  siphon 

10  Op.  cit,  p.  128.  n  Ibid.,  p.  U9.  12  Op.  cit.,  p.  50. 

13  Herzig  and  Ueyee,  Monatshefteiar  Chemie,  Vol.  XV,  p.  613. 

97 


The  Lbcithans 


at  H,  to  enter  the  condenser  D  and  keep  it  at  tlie  proper  temperature.  By  regulating 
the  flow  of  the  water  and  the  height  of  the  flame,  the  temperature  of  the  water  can 
easily  be  kept  within  the  required  limits.  F  are  Geissler  bulbs  for  absorbing  every- 
thing but  the  methyl  iodide,  which  is  absorbed  in  K  and  L.  The  analysis  is  carried 
on  as  follows:  0.2  g.  of  the  substance  to  be  analyzed  is  placed  in  (a)  with  2  g.  of  dry 
ammoniun  iodide  and  enough  hydriodic  acid  (sp.  gr.  1.6)  to  half  fill  the  lower  bulb. 
In  (6)  is  placed  1  g.  of  ammonium  iodide.  The  part  A  of  the  sand  bath  is  filled  with 
sand  and  a  thermometer  reading  to  360°  C.  placed  in  the  sand.  A  stream  of  dry  COg 
is  allowed  to  enter  at  M,  and  when  all  the  air  is  displaced,  a  triple  burner  is  lighted 
under  the  sand  bath.  In  the  meanwhile  the  water  must  be  started  and  kept  running 
through  the  condenser  D  at  a  temperature  of  from  40°  to  50°  C.  As  the  temperature 
of  200°  C.  is  reached  in  the  sand  bath,  methyl  iodide  begins  to  split  off  and  is  carried 
over  by  the  COg  mixed  with  hydriodic  acid  and  iodine.  Most  of  the  iodine  and 
hydriodic  acid  is  condensed  at  D  and  collected  in  C.  Some  passes  over  and  is  absorbed 
in  F,  which  contains  the  following  solution : 

Sodimn  carbonate         -        .        .        .        \  -paxi 

Potassium  arsenite 1  part 

Water 10  parts 

The  methyl  iodide  passes  on  and  is  collected  in  K,  which  contains  2  g.  of  silver 
nitrate  dissolved  in  5  c.c.  water  and  45  c.c.  absolute  alcohol.  The  methyl  iodide  dissolves 
in  the  alcohol,  and  is  decomposed  by  the  silver  nitrate  with  the  formation  of  silver 
iodide.  After  some  time  the  temperature  in  the  sand  bath  gradually  rises  to  240°  C, 
and  after  a  little  while  longer  methyl  iodide  ceases  to  come  over,  as  can  be  seen  by  the 
liquid  in  -K" becoming  perfectly  clear.  L,  which  also  contains  silver  nitrate,  is  used  merely 
as  a  guard.  The  solution  can  be  removed  at  this  point,  and  the  silver  iodide  collected 
corresponds  to  all  the  kephalin  and  one  methyl  group  of  the  lecithin.  Fresh  silver 
nitrate  is  placed  in  K,  another  burner  placed  under  the  sand  bath,  and  the  temperature 
raised  to  300°  0.  The  remaining  two  methyl  groups  of  lecithin  come  over,  while 
kephalin  gives  off  no  more,  or  only  a  trace,  of  methyl  iodide.  The  second  part  of  the 
sand  bath,  B,  is  now  filled  with  sand  and  heated  to  800°  to  decompose  anything  which 
may  have  escaped  previous  heating.  The  two  alcoholic  solutions  containing  the 
silver  iodide  are  diluted  with  much  water  and  warmed  for  several  hours  on  a  steam 
bath  to  remove  alcohol.  Strong  nitric  acid  is  then  added,  and  the  silver  iodide  filtered 
into  a  Gooch  crucible  and  weighed.  In  case  we  are  not  dealing  with  a  mixture  of 
lecithans,  all  the  silver  iodide  can  be  weighed  in  one  portion. 

PEEPAEATION    AND    ANALYSES    OF    VAEIOUS    LECITHANS 

Egg  lecitMn. — The  yolks  of  ten  eggs  are  allowed  to  stand  with  600  c.c.  ether  over 
night,  1  liter  alcohol  added,  the  solution  filtered  and  evaporated  on  water  bath.  The 
residue  is  dissolved  in  200  c.c.  cold  ether  and  1  liter  acetone  added.     The  precipitated 

98 


Waldemar  Kooh  9 


lecithin  is  treated  over  night  with  1  liter  cold  alcohol,  the  solution  filtered  and  evapo- 
rated. The  residue  is  once  more  dissolved  in  ether,  precipitated  with  acetone,  and 
dried  over  sulphuric  acid  in  a  vaccuum  desiccator. 

I."  0.8113  g.  of  the  substance  gave  0.1136  g.  MgoPaO,;  i.  e.,  3.91  per  cent.  P. 

II.  0.9150  g.  of  the  substance  gave  0.1278  g.  MgaPzO,;  i.  e.,  3.90  per  cent.  P. 

III.  0.330  g.  of  the  substance  gave  0.3001  g.  Agl;  i.  e.,  5.80  per  cent.  CH3. 

IV.  0.325  g.  of  the  substance  gave  0.2960  g.  Agl;  i.  e.,  5.81  per  cent.  CH3. 

V.  0.320 g.  of  the  substance  gave,  below  240°  C,  0.0760 g.  Agl;  i.  e.,  153  pei 
cent.  CII3.  The  methyl  iodide  given  off  above  that  temperature  was  lost  on  account 
of  an  accident.  In  III  and  IV  the  methyl  iodide  came  over  at  220°  C.  and  300°  C. 
The  two  portions  were  not  separated. 


*hosphorus  as  3.97 
calculated  for 

III 

Found 

rv 

V 

3CH3:  5.66 

5.80 

5.81 

1 CH3:  1.88 

1.53 

Brain  lecithin  and  kephalin.  —  One  kilo  sheep's  brains  is  minced  in  a  meat- 
chopper and  freed  from  water  and  extractives  by  boiling  with  1  kilo  acetone  for  eight 
hours.  The  solution  is  filtered  cold,  and  the  remaining  acetone  removed  from  the 
brains  by  gentle  heating  at  50°  C.  Seven  hundred  c.c.  cold  ether  are  now  added  and 
allowed  to  stand  for  three  days,  the  solution  is  filtered,  and  another  portion  of  ether 
added,  and  again  allowed  to  stand.  The  ether  filtrates  are  united  and  slowly  evapo- 
rated to  one-fourth  their  original  volume  in  a  tall  beaker.  The  solution  is  then  care- 
fully removed  by  means  of  a  pipette  from  the  white  precipitate,  which  has  settled  to 
the  bottom,  and  1.5  kilo  alcohol  is  added  to  the  solution. 

Kephalin. — The  precipitated  kephalin  is  extracted  five  times  with  boiling  alcohol, 
dissolved  in  ether,  precipitated  with  acetone,  again  dissolved  in  ether,  and  allowed  to 
settle  in  a  long,  narrow,  closed  test-tube.  The  clear  ether  solution  is  removed  by 
decantation,  evaporated,  and  the  residue  recrystallized  twice  from  hot  acetic  ether. 
The  resulting  kephalin  is  very  hygroscopic  and  must  be  dried  over  sulphuric  acid  for 
analysis.  It  agrees  perfectly  in  all  its  properties  with  the  kephalin  described  by 
Thudichum  (p.  127).     On  analysis  it  gave  the  following  results: 

I.  0.2469  g.  of  the  substance  gave  0.5388  g.  CO,  and  0.2159  g.  H2O;  i.  e.,  59.5  per 
cent.  C  and  9.7  per  cent.  H. 

II.  0.415  g.  of  the  substance  neutralized  5.2  c.c.  yV  acid;  i.  e.,  1.78  per  cent.  N. 
Ill  0.9644  g.  of  the  substance  gave  0.1330 g.  Mg^PoO-,;  i.  e.,  3.85  per  cent.  P. 

IV.  0.7235  g.  of  the  substance  gave  0.0990  g.  MgoP^O-,;  i.  e.,  3.82  per  cent.  P. 

V.  0.3488  g.  of  the  substance  gave,  below  240°  C,  0.0945  g.  Agl;  /.  e.,  1.73  per 
cent.  CH3. 

-  VI.  0.490  g.  of  the  substance  gave,  below  240°  C,  0.1312  g.  Agl;  ;".  c.  1.71  per 
cent.  CH3. 

i*For  phosphorus  determinations  the  very  excellent  method  of  Neumann  was  used  (Eiije/manii'j  ^rc/iit'  /<lr 
Physiologie,  1900,  p.  159). 

99 


10  The  Lecithans 


VII.  0.225  g.  of  the  substance  gave,  below  300°  C,  0.0636  g.  Agl;  i.  e.,  1.80  per 
cent.  CH3. 

VIII.  0.748  g.  of  the  substance  were  burned  with  a  mixture  of  nitric  and  sulphuric 
acid,  the  solution  diluted,  neutralized  with  50  g.  barium  hydrate  to  remove  sulphates 
and  phosphates,  the  barium  removed  with  sulphuric  acid,  and  the  solution  evaporated. 
The  ignited  residue  weighed  0.030  g.  Some  of  this  must  have  come  as  an  impurity 
from  the  barium  hydrate.  In  any  case,  the  amount  is  not  sufficient  to  account  for 
any  more  than  an  impurity.  Thudichum  ^^  has  also  found  his  kephalin  to  contain  small 
amounts  of  inorganic  substances  as  impurities.  My  analyses  agree  fairly  well  with 
Thudichum's  (p.  132): 

Thudichum:  C,  60.0;  H,  9.38;  N,  1.68;  P,  4.27 
My  results:    C,  59.5;  H,  9.7;    N,  1.78;  P,  3.84 

The  methyl  groups  correspond  very  closely  to  one  methyl  for  one  nitrogen.  That 
this  is  not  due  to  the  presence  of  lecithin  as  an  impurity  is  shown  by  the  fact  that  the 
methyl  is  all  split  off  below  240°  C. 


Phosphorus  as  3.83 

Found 

calculated  for 

II 

V                     VI 

VII 

1  CH3:     1.85 

1.73            1.71 

1.80 

1  N:         1.73 

1.78 

Lecithin. — The  original  alcohol-ether  filtrate  from  which  the  kephalin  has  been 
removed  by  filtration  is  evaporated  to  dryness,  and  the  residue  dissolved  in  ether  and 
freed  from  cholesterin  by  precipitation  with  acetone.  The  precipitated  lecithin  is 
treated  with  a  large  quantity  of  cold  alcohol  for  some  time,  and  the  solution  filtered 
and  evaporated.  The  resulting  lecithin  should  dissolve  readily  and  completely  in 
cold  alcohol  or  ether.  If  this  is  not  the  case,  the  extraction  with  cold  alcohol  must  be 
repeated.  Finally,  the  lecithin  is  dissolved  in  hot  acetic  ether  and  allowed  to  separate 
out  by  cooling,  dried  over  sulphuric  acid,  and  analyzed. 

I.  0.2420  g.  of  the  substance  gave  0.5683  g.  COg  and  0.2420  g.  HgO;  i.  e.,  64.04 
per  cent.  C. ;  10.4  H. 

II.  0.942  g.  of  the  substance  neutralized  12.1  c.c.  y\j-  acid;  i.  e.,  1.8  per  cent  N. 

III.  0.677  g.  of  the  substance  gave  0.0919  g.  Mg^PgO, ;  i.  e.,  3.79  per  cent.  P. 

IV.  0.815  g.  of  the  substance  gave  0.1109  g.  Mg^PgO, ;  i.  e.,  3.80  per  cent.  P. 

V.  0.3975  g.  of  the  substance  gave  0.3156  g.  Agl;  i.  e.,  5.1  per  cent.  CHg. 

VI.  0.235  g.  of  the  substance  gave  0.1853  g.  Agl;  i.  e.,  5.03  per  cent.  CH3. 

My  lecithin  agrees  fairly  well  in  its  properties  with  the  one  isolated  by  Thudichum 
(p.  116).  The  results  of  the  methyl-group  determination  show  as  good  an  agreement 
with  the  theory  as  can  be  expected  from  a  compound  so  difficult  to  purify. 

Phosphorus  as  3.8 


16  Op.  cit.,  p.  130. 


calculated  for  III  V  VI 

8  CH3:    5.51  5.1  5.03 

1  N:         1.72  1.8 

100 


Waldemab  Kooh  11 


Lecifhans  from  yeast. — Twenty  yeast  cakes  are  mixed  well  with  one  liter  of  alcohol 
in  a  flask  and  boiled  for  eight  hours,  filtered,  the  yeast  allowed  to  stand  with  ether 
over  night,  filtered,  the  filtrates  united  and  evaporated.  The  residue  is  extracted  with 
cold  ether,  three  times  the  volume  of  acetone  added,  and  the  resulting  precipitate  dried 
in  vacuo  over  sulphuric  acid,  as  it  is  not  sufficient  for  further  purification.  The  sub- 
stance thus  obtained  resembles  kephalin  in  that  it  darkens  rapidly  on  exposure  to  the 
air  and  is  precipitated  from  ether  solution  by  alcohol.  The  analysis  gave  the  follow- 
ing result : 

I.  0.220  g.  of  the  substance  gave  0.0286  g.  Mg^PgO, ;  i.  e.,  3.63  per  cent.  P. 

II.  0.165  g.  of  the  substance  gave  0.0626  g.  Agl;  i.  e.,  2.42  per  cent.  CH3. 

The  methyl  iodide  was  split  up  mostly  at  240°  C,  but  some  more  was  obtained  on 
raising  the  temperature  to  300°  C. 

Phosphorus  as  3.63  Fonnd 

calculated  for  II 

1  CH3:     1.76  2.42 

2  CH3:     3.51 

The  amount  of  methyl  is  not  sufficient  to  account  for  two  groups,  and  as  the  com- 
pound was  not  especially  freed  from  lecithin,  it  seems  reasonable  to  account  for  the 
extra  methyl  as  due  to  an  impurity  of  lecithin  which  would  amount  to  about  11  per 
cent.  The  main  bulk  of  the  body  is,  therefore,  kephalin,  which  agrees  well  with  the 
other  observations. 

The  results  so  far  obtained  with  the  Herzig  and  Meyer  methyl  group  determine, 
then,  that  the  two  principal  classes  of  lecithans  differ,  not  only  in  the  fatty  acid  group, 
but  also  in  the  complex  which  contains  the  nitrogen.  Very  striking  is  the  fact  that 
kephalin  occurs  only  in  living  cells,  such  as  the  nerve  or  yeast  cell,  and  is  not  found 
in  the  egg,  which  consists  mostly  of  stored  food  material.  Kephalin  may  possibly  be 
an  intermediary  product  in  the  decomposition  of  lecithin.  The  low  amount  of  carbon 
and  the  correspondingly  large  amount  of  oxygen  would  indicate  an  addition  of  oxygen 
in  the  oleic-acid  radical  of  the  lecithin—  a  hypothesis  which  leaves  unexplained  the  fact 
that  kephalin  is,  if  anything,  more  unsaturated  than  lecithin,  judging  by  the  relative 
amounts  of  iodine  absorbed.  At  any  rate,  there  is  at  present  not  enough  known  about 
the  nature  of  kephalinic  acid  to  trace  its  origin  to  oleic  acid.  As  far  as  the  nitrogen 
complex  is  concerned,  it  is  possible  that  two  methyl  groups  have  been  split  off,  leaving 
a  mono-methyl  oxsethylamin.  Thudichum '"  has,  indeed,  isolated  from  his  kephalin  a 
base  which  contains  less  methyl  groups,  but  he  has  also  mentioned  the  presence  of 
neurin.  My  results,  however,  show  conclusively  that  neurin  can  be  present  only  as  an 
impurity.  The  decomposition  of  twenty  grams  of  kephalin  with  barium  hydrate  yielded 
only  0.2  g.  of  a  platinum  salt,  which  corresponded,  on  analysis,  to  something  between 
a  mono-  or  a  di-methyl  oxrethylamin.  The  investigation  of  this  interesting  relation  will 
be  continued,  and  the  methyl-group  determination  described  above  will  undoubtedly  be 
of  value  in  following  out  the  quantitative  relation  of  lecithin  to  kephalin  under  various 

"  Op.  cit.,  p.  147. 


101 


12  The  Leoithans 


conditions  in  the  living  cell.  The  other  lecithans,  such  as  the  myelins,  paramyelins, 
and  amidomyelins,  isolated  by  Thudichum  from  brain  tissues,  do  not  seem  to  occur  in 
any  large  quantity  and  have  not  as  yet  been  investigated. 

PHYSIOLOGICAL    PROPEETIES 

Substances  of  such  importance  to  the  cell  as  the  lecithans  must  possess  some  valne 
as  foods.  The  action  of  the  digestive  ferments  is  therefore  of  especial  importance, 
as  neurin,  which  is  formed  by  the  decomposition,  has  been  shown  by  Halliburton" 
to  have  a  decided  efPect  on  blood  pressure.  A.  B6kay,'*  under  the  direction  of  Hoppe 
Seyler,  investigated  this  problem  and  found  that  lipase  will  split  egg  lecithin  into 
glycerophosphoric  acid,  fatty  acids,  and  neurin.  The  three  splitting  products  must, 
however,  be  immediately  absorbed  and  resynthesized,  as  they  are  not  found  in  the 
urine  or  faeces,  and  a  meal  containing  considerable  lecithin  has  never  been  known  to 
cause  any  bad  effects.  The  results  on  the  metabolism  of  the  injection  of  lecithin 
into  the  circulation  are  as  yet  in  too  unsatisfactory  a  state  to  be  discussed.  The 
patenting"  of  brain  preparations  for  medical  purposes  seems,  therefore,  especially 
premature. 

For  completeness'  sake  a  number  of  facts  have  been  mentioned  in  this  paper 
which  have  been  known  for  a  long  time.     The  new  facts  are  as  follows : 

1.  The  word  "lecithan"  is  to  be  used  as  a  group  name,  including  such  com- 
pounds as  egg  lecithin,  brain  lecithin,  kephalin,  myelin,  paramyelin,  etc. 

2.  The  emulsion  formed  by  the  lecithans  may  be  the  substratum  in  which  the 
reactions  of  the  cell  take  place.  The  precipitation  of  this  emulsion  by  divalent  kations 
is  prevented  by  univalent  and  trivalent  kations,  as  far  as  investigated,  and  this  obser- 
vation may  furnish  an  explanation  of  the  changes  brought  about  by  electrolytes  in 
the  living  cell. 

3.  Kephalin  is  found  only  in  the  living  cell,  and  may  be  an  intermediary  product 
in  the  metabolism  of  lecithin. 

4.  An  accurate  method  for  determining  lecithin  and  kephalin  quantitatively. 

"  Op.  cit,  p.  55.  19  C.  Zeebe,  Chemiker-Zeitung  (1902),  Vol.  XXVI,  p.  17s 

18  A.  B6KAY,  Hoppe  Seyler's  ZeiUchrift  filr  physiolo-       Deutsches  Reichs-Patent,  127357. 
gische  Chemie  (1877),  Vol.  I,  p.  157. 


102 


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